1. Field of the Invention
The present invention relates to methods of improving the introduction of DNA into bacterial host cells.
2. Description of the Related Art
Type II restriction endonucleases are reportedly effective barriers to the introduction of DNA into bacteria (Briggs et al., 1994, Applied and Environmental Microbiology 60: 2006-2010; Accetto et al., 2005, FEMS Microbiology Letters 247: 177-183). Numerous Type II restriction endonucleases have been characterized in Bacillus and many commercially available restriction endonucleases have been isolated from Bacillus species (Roberts, et al., 2005, Nucleic Acids Research 33: 230-232).
Host DNA is protected from cleavage by its native restriction endonuclease due to host DNA modification by a corresponding DNA methyltransferase. The restriction endonuclease and DNA methyltransferase genes usually lie adjacent to each other in the genome and constitute a restriction-modification (R-M) system. These genes may be oriented transcriptionally in a convergent, divergent, or sequential manner. Although restriction endonucleases have little if any sequence similarity between one another, a limited amino acid motif, PD . . . D/EXK, has been found in many restriction endonucleases (Pingoud and Jeltsch, 2001, Nucleic Acids Research 29: 3705-3727). In contrast, several general motifs have been found for the DNA methyltransferases (Kumar et al., 1994, Nucleic Acids Research 22: 1-10; Smith et al., 1990, Proceedings of the National Academy of Sciences USA 87: 826-830), which has allowed identification of restriction endonucleases by first identifying their more homologous corresponding DNA methyltransferases.
The introduction of DNA into a bacterial host cell, e.g., Bacillus licheniformis, can be an inefficient process, resulting in few, if any, transformants. There is a need in the art for new methods of introducing a DNA into a bacterial host cell to improve the efficiency of obtaining transformants with the DNA.
The present invention relates to improved methods of introducing DNA into a bacterial host cell.